Urinary tract infections (UTI) are highly prevalent infections that have an increasing propensity for recurrence/and or chronicity and antibiotic resistance. Nearly 50% of women will experience a UTI during their lifetime with 20-40% of these women will suffer from multiple recurrent infections (rUTI), resulting in serious deterioration of their quality of life including: pain, discomfort, disruption of daily activities,and few treatment options other than long-term antibiotic prophylaxis. Over 1 million women are referred to urology offices every year because of troubling rUTI. The high rate of UTI and rUTI highlights the need for a better understanding of the life cycle of the most common causative agent of UTI, uropathogenic E. coli (UPEC). The source of UPEC that leads to a majority of UTIs is the gut microbiota. When present in the gut, UPEC are shed in the feces and may then colonize and ascend the urethra to infect the bladder. Understanding the mechanisms that contribute to acute UTI and progression to rUTI is of critical importance to develop new therapeutics. Since UPEC that lead to UTI reside in the gut, this includes understanding the factors governing the residence of UPEC in the intestines. To colonize the host, UPEC express a family of adhesive fibers termed chaperone usher pathway (CUP) pili. I have established the first described role for two CUP pili, type 1 and F17-like, in mediating UPEC intestinal colonization. The proposed work in this grant aims to: 1) define the ligand for F17-like pili and determine how the expression of these pili, during healthy states and periods of inflammation, influences UPEC intestinal tropism; and 2) to identify small-molecule pilus inhibitors that target type 1 and F17-like expression in vitro and in vivo as well as use these inhibitors to create a combinatorial treatment that eradicates UPEC from the gut and bladder. These aims will be completed using established biochemical and genetic techniques. As well as state-of-the-art gnotobiotic techniques including examining UPEC colonization in mice containing synthetic, defined microbiota communities. Overall, the findings from this grant will provide novel information regarding UPEC colonization of the source niche, the gut, including: i) elucidating the molecular mechanism by which CUP pili influence UPEC intestinal colonization, ii) determining how alterations in intestinal inflammation influence UPEC colonization, and iii) how targeting CUP pili that enhance UPEC intestinal colonization effects overall colonization ability. The application of the research training program set forth by Dr. Scott Hultgren, the sponsor, and Washington University, the sponsor institution, will allow for the completion of the research goals set forth above. This research training program includes: relevant course- work in molecular microbiology, immunology, and genetics; opportunities to present and receive feedback on research directions and results at numerous seminars; hands-on experimental guidance from collaborators to learn additional relevant techniques; and meetings once a week with the sponsor to examine and discuss the progression of training and research goals.